Several approaches have been taken to improve gene transfer into hematopoietic cells of non-human primates. These include the use of novel retroviral vectors, the use of alternative viral vectors, and the use of hematopoietic growth factors to expand the numbers of CD34+ cells in the peripheral blood (PB) and bone marrow (BM). Over the past year emphasis has been placed on the use of retroviral vectors that utilize the Moloney murine leukemia virus (MoLV) envelope protein, the use of retroviral vectors that differ in the viral envelope protein used (such as that of the gibbon ape leukemia virus (GaLV)), and the use of non-retroviral vectors (such as the adeno-associated virus (AAV)). By altering the envelope protein used by the vector or the vector itself, it is hoped that the tropism of the vector would be broadened to include not only other cell types, but non-dividing cells as well. Based on in vitro and in vivo studies, we found the tropism of the GaLV vector to be more restricted than that of MoLV based vectors. Although the GaLV vector was highly successful in targeting CD4-enriched lymphocytes, it was not as effective in targeting immunoselected CD34+ cells. In fact, the GaLV receptor was found to be expressed on a very restricted subset of CD34+ cells that were small in size and Thy-1+. An AAV vector initially appeared to be just as successful in targeting CD34+ cells in vivo as that of MoLV based retroviral vectors. However, unlike MoLV based vectors, the AAV vector could only be detected transiently in circulating leukocytes. In an attempt to further improve gene transfer efficiencies, cytokine mobilized CD34+ cells were collected and transduced. For these studies, non-human primates received granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) for 4-5 days. Cytokine stimulated CD34+ cells were immunoselected from PB leukocytes collected by a novel leukapheresis procedure and/or from BM. The immunoselected CD34+ cells were then cultured and transduced with MoLV based vectors. The use of cytokine mobilized CD34+ cells were found to improve hematopoietic reconstitution following myeloablation. In addition, gene transfer efficiencies were improved, with approximately 5% of circulating leukocytes containing provirus. With continued improvements in vector design, and our understanding of stem cell biology and gene regulation, primate models will help in developing safe and effective gene transfer protocols for human subjects.